The success of a broad array of microbial pathogens, from viruses to bacteria to eukaryotic parasites, depends on the ability to gain entry into and/or transport proteins into the cytosol of host cells. Intracellular acting bacterial toxins have evolved to take advantage of numerous host-mediated entry mechanisms {Knodler, 2001}, and therefore, these toxins are ideal tools for studying endocytosis and vesicular trafficking. Indeed, the use of bacterial toxins has led to many key discoveries including, but not limited to, membrane recycling, clathrin-independent endocytosis, and retrograde transport {Sandvig, 2005}. Small molecules that disrupt toxin binding, entry, trafficking and host-response can not only serve as novel probes to dissect such eukaryotic cellular pathways, but are also possible therapies for infectious and genetic diseases. Several groups have identified compounds that inhibit entry of ricin, Shiga toxin, and Pseudomonas aeruginosa exotoxin A (ExoA) into host cells. (Saenz, 2007, Wahome, 2010, Stechmann, 2010). These small molecules exhibited varied mechanisms of action, including blockage of retrograde toxin trafficking at the early endosome-TGN junction, morphological disruption of the Golgi apparatus, and inhibition of the toxin active site.
Bacillus anthracis, the causative agent of the disease anthrax, secretes binary toxins that enter host cells and disrupt physiological processes. Lethal factor (LF) is a Zn2+-dependent metalloprotease that cleaves mitogen activated protein kinase kinases (MKKs) 1-4, 6, and 7 {Duesbery, 1998; Vitale, 1999} and Nlrplb and reproduces many pathologies of anthrax when injected into laboratory animals {Beall, 1962, Moayeri, 2009}. The cellular entry of LF is dependent on a cell-binding and translocation subunit known as protective antigen (PA). PA is an 83 kDa protein that is cleaved by host proteases into 63 and 20 kDa fragments, allowing oligomerization of the toxin into a prepore (Milne, 1994). The PA oligomer can then bind up to four monomers of LF, forming a holotoxin complex (Mogridge, 2002, Kintzer, 2009). Two cellular toxin receptors, TEM8 and CMG2, mediate toxin binding via a structural domain and interaction motif homologous to those seen in integrin binding {Bradley, 2001, Scobie, 2003, Bradley, 2003} and endocytic uptake. Also similar to integrins, PA binding to receptors is modulated by interactions between the cytosolic tail of the receptor and the actin cytoskeleton (Go, 2009, Garlick, 2010). Palmitoylation of cysteines in the cytosolic tail of the receptor is important to prevent premature clustering, internalization, and lysosomal degradation (Abrami, 2006). Upon toxin binding, oligomerized receptors migrate into detergent-resistant membrane fractions known as lipid rafts, promoting recruitment of the E3 ubiquitin ligase Cb1 by beta-arrestin and ubiquitination of the receptor (Abrami, 2003, Abrami et al. 2006). The receptor-toxin complex then recruits the adaptor protein AP-1 and clathrin. The newly formed clathrin-coated vesicle pinches off from the membrane in a manner dependent on actin and dynamin and transits to the early/sorting endosome (Abrami, 2010, Abrami, 2003). ARAP3 (ArfGAP and RhoGAP with ankyrin repeat and PH domains) also plays a role in toxin endocytosis, though its precise contribution is unclear (Lu, 2004). Acidification of the lumen of the late endosome drives a conformational change in the prepore resulting in insertion into the endosomal membrane and translocation of LF into the cytosol (Koehler, 1991; Milne, 1993; Miller, 1999). Alternatively, LF may be translocated to the interior of intraluminal vesicles and transported to the late endosome via multivesicular bodies in a process dependent on COPI and ALIX (Abrami, 2004). The vesicular membranes then fuse with the limiting endosomal membrane and thereby deliver LF to the cytosol (Abrami, 2004).
Small-molecules that interfere with acidification of cell transport vesicles called endosomes have been shown to have multiple effects in human health and disease. Lysosomotropic agents, which preferentially accumulate in cell-trafficking vesicles and raise pH, inhibit the effects of bacterial toxins such as anthrax toxin and Diphtheria toxin and limit the survival of viruses (Ooi, 2006, Perez 1994, Guinea, 1995). The compound chloroquine has been investigated as a potential therapeutic in HIV infection (Romanelli, 2004). Bafilomycin A1, a compound which specifically inhibits the vacuolar ATPase resulting in increased endosomal pH, has also been investigated for use in bone disorders involving excessive resorption (Sorensen, 2007, Niikura, 2005) and/or tumor treatment (McSheehy, 2003). It has been proved in principle that toxin inhibitors can partially protect animals from B. anthracis infection alone and can extend therapeutic window when used in conjunction with antibiotics (Shoop, 2005). Several small-molecules which disrupt anthrax toxin transport by raising endosomal pH have been discovered and shown to block intoxication both in cell-based in vitro systems and in vivo in animals. Anthrax toxin killing of cells is inhibited by compounds that inhibit acidification of the endosome such as ammonium chloride, chloroquine, and monensin (Friedlander, 1986). Chloroquine improves survival in a mouse intoxication model (Artenstein. 2004). Quinacrine, a molecule which accumulates in acidic endosomes and effectively raises endosomal pH, protects cells from LT, but was unable to protect animals in a spore infection model of disease (Comer, 2006). Several well-studied compounds with diverse known mechanisms of action have also been shown to inhibit intoxication by raising endosomal pH, rather than by the previously known action of the compound. The Ca2+ channel inhibitors amiodarone and bepridil, the P2X7 antagonist o-ATP, the natural product diphyllin, and the teniacide niclosamide all inhibit LT through disruption of proton gradients (Sanchez, 2007, Moayeri, 2006, Zhu, 2009).
The present invention addresses these and other problems in the prior art.